In this FY, we accomplished and advanced several projects. They are (1) Identifications of unique properties of parasite-induced membranes, including vacuole membrane (PVM) and Maurers Cleft, and differences in properties of parasitized erythrocyte membranes between HbA and HbS erythrocytes, (2) Direct visualizations of whole MC by electron microscopy, (3) Microscopy study of gametocyte metabolism and drug resistance relationship, and (4) Identifications of hemoglobin distributions and their modifications using hyperspectral imaging combined with optical phantom. (1) We continued studying parasite-derived membranes with unique biophysical properties using fluorescence lifetime microscopy (FLIM). During invasion process, PVM surrounds invading parasite. PVM continuously increase in its size as parasites mature. In addition, new membranes emerge from PVM and form Maurer's cleft (MC). This structure seems to play crucial roles in trafficking and delivery processes for parasite-derived proteins. However, the source of lipids enough to support the size increase in the new membrane structure is not known. A key to solve this question is cholesterol content as parasite does not have a cholesterol synthesis mechanism. In addition, cholesterol is one of the most important molecules for membrane biophysical properties. The potentially unique properties of PVM and MC could provide important insights for their functions responsible for efficient trafficking of parasite proteins to erythrocyte membrane. In previous FYs, we identified the lipid exchange/transfer between parasite and host erythrocyte membranes during intraerythrocytic stages monitored by the translocations of membrane dye, Di4-ANEPPDHQ, and cholesterol analogue, bodipy-cholesterol. These results suggest that lipids are transferred between erythrocyte and parasites, and transfer mechanism may not be the same between cholesterol and non-sterol lipids such as phospholipids. We also found that hemoglobin S erythrocytes, both heterozygous (AS) and homozygous (SS) non-parasitized erythrocytes, had lower Di-4 lifetime compared to non-parasitized HbA erythrocytes. Differences in Di-4 lifetime were also observed in parasitized erythrocytes while the same trend of lifetime differences between erythrocyte, PV and parasite membranes within a same genotype. These data indicate that HbS erythrocytes have significantly different membrane property than that of normal HbA erythrocytes, which may control protein assembly and distributions on the host erythrocyte membrane. In this FY, we found that that cholesterol gradient inside parasitized erythrocytes are created by constant flow of non-sterol parasite lipids from parasite to PVM and small flux of cholesterol to PVM from surrounding host erythrocyte membrane to compensate exceeding reduction of relative cholesterol level at PVM. (2) We continued studying gametocyte metabolism using microscopy approach. In previous FY, we identified (a) a high lipid content, but similar cholesterol level, (b) reduced reactive oxygen species, and (d) similar mitochondrial membrane potential. We also studied requirement of mitochondrial activity for gametocyte developments and found complex physiological variability exist in gametocyte developments. In this FY, we focused on studying level of metabolic products on lipid metabolism and potential gluconeogenesis. Using fluorescence indicators, we are currently measuring (a) glycerol, (b) fatty acid, and (c) triacylglycerides and comparing these levels with asexual stages of parasites to study relative activities of each metabolic cascade. Using neutral lipids indictor, we found that neutral lipids in gametocytes are stored in droplet-like compartments along with the parasite plasma membrane. This distribution is very different from asexual stages of parasite in which neutral lipids distribute diffusedly in the parasite with some concentrated areas. These data should provide important information on the mechanism of gametocytogenesis and following sexual stage. (3) In previous FY, we initiated a project studying hemoglobin distributions within a erythrocyte using hyperspectral imaging. Hemoglobin molecules have specific absorption spectra with several characteristic peaks, which change to different spectra as hemoglobin degrades. Hyperspectral imaging uses a specially designed optics and changes wavelength of incident light, and scans from UV to near IR regions. Intensities of captured images at each wavelength were compared to original light intensities and absorption spectra were produced. This technique has multiple advantages as absorption spectra of any area of cell can be analyzed that potentially help estimating hemoglobin amount and the level of degradations. We established technical analysis protocol to estimate hemoglobin concentration in terms of hyperspectral imaging signals. We published this work to J. of Selected Topics in Quantum Electronics. This FY, we used optical phantom with confined hemoglobin solution with triangular prism shape. Known dimension of hemoglobin solution provide accurate information of optical absorbance data with respect to estimated volume of hemoglobin solution. We are now preparing a manuscript for this work.